AB0085 CYTOKINE DRIVEN ECM PRODUCTION IS HIGHLY DEPENDENT ON DISEASE DRIVERS IN FIBROBLAST-LIKE SYNOVIOCYTES
Authors: S. F. Madsen, D. Sinkeviciute, M. Karsdal, A. C. Bay-Jensen, C. Thudium
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Fibrosis is a significant component in arthritis diseases that involves the joint. It can lead to joint swelling, stiffness and ultimately to pain. It is the synovial fibroblasts which secrete the extracellular matrix (ECM) components, and upon activation the production increases. The healthy synovial membrane consists of multiple ECM components, including type I, III and VI collagen<sup>[1]</sup>. The ECM composition of the fibrotic and fibro-inflamed membrane is unclear. We have previously shown that TGF-β increases the formation of type I, III and VI collagens in osteoarthritis (OA) fibroblasts-like synoviocytes (FLS)<sup>[2]</sup>. Both IL-1 and TNFα receptors have been found in the synovium of rheumatoid arthritis (RA) patients<sup>[1]</sup>. We hypothesize that both fibrosis and inflammation are likely to be present in arthritis diseases at the same time. And that the interplay between different disease drivers is crucial for the structural manifestation. Therefore, the combination of both growth factors and cytokines may be directing the composition of the synovial ECM in the joint. <h3>Objectives</h3> The objective of this study was to characterize the effect of inflammation on the fibrotic response of OA FLS using biomarkers of collagen formation. <h3>Methods</h3> Primary human FLS were isolated from synovial membranes, from OA patients undergoing total knee replacement (n=4) and cultured in the Scar-in-a-Jar model. Briefly, the fibroblasts were cultured in 0.4% fetal bovine serum DMEM, ficoll (to create a crowded environment) and ascorbic acid for 12 days. The fibroblasts were stimulated with TGF-β1 [1 nM], IL-1β [0.5 nM] or TNF-α [0.5 nM], either alone or in combination from day 0 and the treatments changed twice a week. Non-stimulated fibroblasts were used as control. Biomarkers of type I, III, and VI collagen formation (PRO-C1, PRO-C3 and PRO-C6, respectively), were measured in the culture supernatants at days 0, 4, 8 and 12 using validated ELISAs. <h3>Results</h3> Treatment with IL-1β or TNF-α alone did not affect PRO-C1, PRO-C3 or PRO-C6, compared control (Figure 1.A). TGF-β1 increased the total release of PRO-C1 and PRO-C6, compared to control (p&lt;0.001, Figure 1.A). The total release of PRO-C3 did not increase, while at day 12 the PRO-C3 level was higher compared to control (p&lt;0.05; data not shown). Both IL-1β and TNF-α in combination with TGF-β1 decreased PRO-C1 (p&lt;0.01, Figure 1.A; decrease of 32-53%, Figure 1.B) and PRO-C6 compared to of TGF-β1 alone (p&lt;0.001, Figure 1.A; decrease of 56-78%, Figure 1.B). TNF-α in combination with TGF-β1, increased the release of PRO-C3 (p&lt;0.01, Figure 1.A; increase of 172-264%, Figure 1.B), while the combination with IL-1β did not seem to affect the PRO-C3 response compared to TGF-β1 alone (Figure 1.A; increase of 48% to decrease of 39%, Figure 1.B). <h3>Conclusion</h3> The combination of inflammatory cytokines and TGF-β1 leads to a differential effect on formation of ECM components such as type III collagen. These findings suggest that the presence of an inflammatory component, may be important for the development of a fibrotic component in joint diseases. Consequently, this also underlines the need for a better understanding of the molecular mechanisms driving the joints diseases. <h3>References</h3> [1]Smith MD. The normal synovium. <i>Open Rheumatol J</i>. 2011;5:100-106. [2]Madsen SF, Madsen SS, Bay-Jensen AC, Thudium CF. Clear differences in extracellular matrix production biomarkers between dermal fibroblasts and osteoarthritis fibroblasts-like synoviocytes. <i>Osteoarthritis and cartilage.</i> 2022; 30: supp 1. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> Sofie Falkenløve Madsen: None declared, Dovile Sinkeviciute Employee of: Nordic Bioscience, Morten Karsdal Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience, Anne-Christine Bay-Jensen Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience, Christian Thudium Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience.